The focus of this research project is the study of sporulation in the bacterium Myxococcus xanthus as cells form multicellular fruiting bodies. Starvation in M. xanthus triggers the developmental program, which causes cells to aggregate and sporulate. In this proposal, we plan to study the regulation of the timing of sporulation, as sporulation normally occurs in raised mounds after the completion of aggregation, but not in unaggregated cells. The delay in sporulation suggests that cells have a mechanism to monitor progress towards aggregation prior to triggering sporulation. The timing of sporulation is an interesting problem since sporulation marks the endpoint of a developmental program and the "decision" to follow that pathway is controlled by the microenvironment of the cells, which includes the spatial location of cells in reference to neighbor cells. The timing of developmentally regulated events in response to cell positioning is important for the proper development of all multicellular or organisms, especially higher eukaryotes. We have recently discovered two genes that appear to involved in the regulation of the timing of sporulation: espA and espB. Mutant phenotypes suggest that EspA inhibits sporulation until aggregation has been completed and EspB counters this inhibition. We propose to study the structure of EspA and how the individual domains modulate the negative regulation of downstream (sporulation) genes and upstream signaling genes. We plan to characterize these domains by preparing and analyzing mutants and studying the effects of the mutations on the functioning of the protein in regulating sporulation. We plan to follow the localization of EspA and EspB during development by cell fractionation studies, fluorescence microscopy, and construction and tracking of GFP fusions. The two Ser/Thr protein kinases encoded by genes flanking espAB will be studied for their possible roles in regulating the interaction between EspA and EspB. We plan to examine EspA and EspB for phosphorylation changes during development. In addition, we plan to characterize HpkA, a histidine protein kinase that regulates the expression of EspA and the substrate for the kinase. We also plan to search for the genes and gene products that are regulated by EspA - the downstream genes that initiate the sporulation process. The last part of the grant will explore the effect of C-signaling and high cell density in the control of sporulation.